IMMUNITY IN PASTEURELLA MULTOCIDA INFECTIONS

V irtually all of the available information relating to immunity in pasteurellosis refers to infections with

P. multocida. However, because of the broad host range of P. multocida and the diversity of animal
models used to study immune responses and immunity, it is diffi cult to draw overall conclusions about
the mechanisms of immunity. Indeed immunity will clearly be different according to the animal species
and the type of infection. It is therefore not possible to extrapolate data obtained from infection with a
specifi c serogroup, serotype, or strain in a particular animal species to other P. multocida infections.

Innate Immunity

Innate host defense mechanisms such as phagocytosis, the production of antimicrobial peptides, and
complement are key components of nonspecifi c natural resistance to infection. However, P. multocida
has evolved mechanisms for circumventing these in the na ï ve host. Bacterial capsules protect bacteria
from phagocytosis and from the bactericidal action of complement. The capsule of P. multocida is
critical in mediating evasion of phagocytosis by macrophages (Boyce and Adler 2000 ). A genetically defi
ned mutant showed enhanced engulfment, thereby confi rming earlier work that showed that P.
multocida was in general resistant to phagocytosis by both macrophages and heterophils unless
opsonized (Harmon et al. 1 992) and that susceptibility to phagocytosis could be increased by enzymatic
removal of capsule with hyaluronidase (Harmon et al. 1 991) . The ability of P. multocida to survive and
grow in the presence of fresh serum was abrogated by mutation of the capsule biosynthesis locus
(Chung et al. 2001 ), consistent with an earlier work that showed a correlation between capsule and
serum resistance (Hansen and Hirsh 1 989) . However, some very low- virulence strains can grow and
multiply in 90% chicken serum (Diallo and Frost 2000 ); clearly other factors are also important for
serum resistance. Antimicrobial peptides produced by the host appear to play at least some role in
innate immunity

to FC- c ausing strains of P. multocida in chickens; there is no information for other serotypes. Wild-
type P. multocida was resistant to the avian cathelicidin peptide fowlicidin, with resistance heavily
dependent on expression of entire, full length LPS (Harper et al. 2 007a 2007b) . Fowlicidin is a cationic
peptide and the charge -b ased interaction of the peptide with the negatively charged phosphate
residues on lipid A is predicted to be an essential initial step in the antimicrobial action (Daugelavicius et
al. 2000 ). P. multocida strains that express LPS lacking the terminal phosphocholine residues are signifi
cantly more susceptible to the action of fowlicidin (Harper et al. 2 007b) . It is likely that the charge on
the phosphocholine residues helps to reduce the interaction of the cationic peptide with lipid A.
However, strains that express LPS lacking the heptose side chain are much more susceptible to killing by
fowlicidin, suggesting that these residues form a steric barrier to the peptide (Boyce et al. 2009 ). E arly
work showing major histocompatibility complex (MHC)- l inked resistance to FC in chickens has not
been investigated further (Lamont et al. 1987 ).

Acquired Immunity

There is long - standing evidence that immunity to

 Pasteurella infection is mediated principally by antibody. Passive immunity can be transferred with
immune serum from mice, rabbits, chickens, turkeys, and cattle. Additionally, immune serum has
opsonic and bactericidal activity (Wijewardana et al. 1 990; Ramdani and Adler 1 991) and anti- L PS
monoclonal antibodies are also opsonic and protective in mice (Wijewardana et al. 1 990) .
Nevertheless, in chickens at least, cellular immunity also appears to be important; protective immunity
could be transferred by immune spleen cells or by culture supernatant from immune splenocytes (Baba
1 984) . Immunity in bovine HS and bovine respiratory pasteurellosis is also presumed to be mainly
antibody - mediated, as immune bovine serum can passively protect mice. However, the actual role of
antibody in bovine immunity is far from clear, since some studies have shown a correlation of antibody
with protection while others have found no relationship (Dabo et al. 2007 ). More work is therefore
required to defi ne the respective roles of humoral and cellular immunity in most species.

ttempts to manipulate the immune response to

P. multocida have included the administration of melatonin to rats; immune modulation was
dependant on the time of day of administration (Korde et al. 2 005) . Enhancement of both antibody
response and protective immunity to P. multocida infection in mice was achieved by DNA - based
delivery of a hybrid Interleukin- 2 gene (Xie et al. 2 007) . FC was one of the fi rst bacterial infections
against which vaccination was attempted. Louis Pasteur famously showed that repeated in vitro
passage of P. multocida resulted in attenuation of the bacteria and that chickens inoculated with the
attenuated strain were then resistant to challenge with a virulent strain. Of course, in Pasteur’ s strain
the basis for attenuation was not known, with reversion to virulence a major potential problem.
Nonetheless, this important work demonstrated the feasibility of immunoprophylaxis. Since then a
range of vaccines has been investigated, mostly for use in cattle and buffalo or poultry. Why then, over
125 years later, do we still not have available an ideal vaccine? Currently each of the available vaccines
has signifi cant drawbacks. In general, bacterins give only short - term, serotype - specifi c immunity,
with sometimes variable effi cacy, and they must be delivered by injection. Conversely, live attenuated
vaccine strains can be administered in food or water, and give better cross - serotype protection, but the
currently available undefi ned, attenuated, strains have caused disease under some circumstances.
While much effort has been focused on the development of a cross- p rotective subunit vaccine, there
are none in current production. As well as vaccine effi cacy, cost of vaccine production and
administration is a critical consideration in vaccine development, as profi t margins for growers are
generally very small.

B acterins and Cell Components

There is general consensus that bacterin vaccines can stimulate immunity in poultry and in cattle, but
that immunity is relatively short - lived and is restricted to homologous or closely related serotypes. This
does not constitute a major problem for HS, where the causative serotypes (B:2 and E:5) are closely
related. Consequently, bacterins against HS have been used with relatively good success rates for over
100 years (De Alwis 1 992; Verma and Jaiswal 1998 ); these vaccines are administered with a range of
adjuvants usually consisting of oil - water
emulsions. The effectiveness of these oil - adjuvant vaccines has been questioned from time to time and
has been evaluated many times, but even a relatively recent study demonstrated effi cacy in buffalo
calves, although the numbers of animals were small (Shah et al. 2001 ). Mice vaccinated with a serotype
B:2,5 vaccine were not protected against challenge with a B:3,4 strain. Vaccination against pneumonic
pasteurellosis presents additional problems (reviewed by Dabo et al. 2 007) , with one study fi nding no
protection in calves vaccinated intratracheally with killed bacteria (Dowling et al. 2 004) . Cross -
protective immunity is relevant in the development of poultry vaccines, where it is clear that laboratory-
c ultured, killed bacterial cells elicit only homologous protection. The reason for this restriction is almost
certainly that important cross p rotective protein antigens are poorly expressed by

P. multocida during some conditions of in vitro growth. Bacteria harvested from infected tissues and
then killed are able to stimulate heterologous immunity (Rebers and Heddleston 1 977) , which can be
transferred by serum to naï v e turkeys (Rebers et al. 1975 ). There is evidence that the cross -
protective antigens may be expressed in vitro under low - iron conditions (Glisson et al. 1 993; Ruffolo
et al. 1 998) . The identity of some of the individual proteins is discussed below.

Live Vaccines

E mpirically derived, live, attenuated strains of P. multocida have been used as vaccines against FC
mainly in turkeys. The original Clemson University (CU) A:3,4 strain stimulates both humoral and cellular
immunity and induces protection when administered in drinking water to turkeys, but not in chickens.
Several derivatives of the CU strain have been developed, including temperature sensitive M- 9 , PM#1,
and PM#3 strains unable to grow at 42 ° C in vitro. However, the mechanism of attenuation for all of
these strains is unknown and the vaccines retain a degree of virulence under experimental conditions
(Hopkins and Olson 1997 ) and have occasionally caused disease outbreaks in the fi eld (Hopkins et al.
1998 ). Live vaccines for HS have been derived from fi eld isolates, such as the serotype B:3,4 strain
isolated from deer (Myint et al. 1987 ). Again, while demonstrating a good degree of effi cacy, the basis
for the observed attenuation is unknown and these vaccines have not gained widespread usage (Verma
and Jaiswal 1 998) .

Genetically defi ned, attenuated strains capable of stimulating protective immunity have been
constructed. Mutation of the aroA gene leads to attenuation of P. multocida due to the inability of the
bacteria to synthesize aromatic amino acids, whose low concentrations in host tissues results in reduced
growth in vivo . The defi ned aroA mutants of A:1 and A:3 FC- c ausing strains stimulate heterologous
protection in both mice (Homchampa et al. 1992 1997 ) and in chickens (Scott et al. 1999 ). Similar
attenuated aroA mutants have also been constructed in a B:2 background strain. Systemic or intranasal
vaccination of mice elicited solid immunity (Tabatabaei et al. 2002 ). Unfortunately, in calves
intramuscular injection of the vaccine was necessary to stimulate protective immunity, with intranasally
vaccinated calves remaining completely susceptible (Hodgson et al. 2 005; Dagleish et al. 2007 ).
Genetically defi ned acapsular mutants are attenuated in both A:1 and B:2 backgrounds (Boyce and
Adler 2001 ; Chung et al. 2005 ), but their potential as vaccines has not been developed further.

Individual Antigens
 The identity of protective antigens in P. multocida infections has remained surprisingly elusive. It is
clear that LPS plays some role in immunity; anti- L PS monoclonal antibodies are opsonic and can
transfer at least partial protection, but immunisation of mice with LPS failed to elicit any protection
(Wijewardana et al. 1990 ; Ramdani and Adler 1991 ). LPS - based immunity is by defi nition restricted
to the homologous serotype. A cross - protective protein antigen of 39 - kDa purifi ed from P. multocida
grown in vivo (Rimler 2001 ) was localized to the bacterial cell surface using a monoclonal antibody,
but not identifi ed (Rimler and Brogden 2001 ). However, the protective antigen was later identifi ed
erroneously as the lipoprotein PlpB (Tabatabai and Zehr 2004 ), which shows similarity to methionine -
binding proteins from many other bacterial species. In fact, the cross - protective antigen is PlpE, a
lipoprotein with homologs in Mannheimia haemolytica and

Actinobacillus pleuropneumoniae that stimulate protective immunity in cattle and pigs (Wu et al. 2
007) . Recombinant soluble PlpE elicited cross- p rotection in mice and chickens; signifi cantly,
immunization with PlpB resulted in no protection

in either mice or chickens (Wu et al. 2007 ). Work in the authors’ laboratory has confi rmed these fi
ndings and also shown that insoluble PlpE is able to stimulate protective immunity in chickens and mice.
T he major outer membrane porin OmpH is likewise able to stimulate protective immunity. Anti- O mpH
monoclonal antibodies protected mice via opsonization (Vasfi Marandi et al. 1996 ). Work in chickens
also showed that OmpH was a protective antigen. Interestingly, native but not recombinant OmpH was
protective, as was a cyclic peptide corresponding to a predicted exposed loop of the protein (Luo et al.
1999 ); other peptides were not active. A subsequent study claimed protection of mice with
recombinant fragments of OmpH (Lee et al. 2 007) , although statistical signifi cance was marginal. No
chicken experiments were performed. Recently, a mutant in the regulatory gene fur was used to
generate outer membranes rich in iron - regulated proteins (including OmpH); vaccination with outer
membranes purifi ed from this mutant stimulated immunity in mice (Garrido et al. 2008 ), although the
numbers of animals were small, thus making statistical analysis diffi cult. Intriguingly, the absence of
OmpH in a second mutant increased the degree of cross protection conferred. O ther vaccine studies
with individual proteins that showed some level of protection included a 37.5- k Da (Lu et al. 1 991) and
a 39- k Da protein (Ali et al. 2004 ; Sthitmatee et al. 2008 ). However, in these studies the identity of
these proteins was not determined and it is very likely that these proteins were actually PlpE. No
protection was seen following immunization of mice with OmpA or turkeys with P6 protein. No vaccines
based on individual protein antigens have been developed commercially. I mmunity against porcine AR
is dependent on antibodies against the dermonecrotic toxin, PMT, elaborated by AR - causing strains of
P. multocida ; disease can be reproduced experimentally with purifi ed toxin. Accordingly, a range of
toxin derivatives has been developed for successful immunization against AR. Approaches have included
formaldehyde - inactivated toxin (Foged et al. 1989 ), the construction of non - toxigenic derivatives by
specifi c site -d irected mutagenesis (S1164A and C1165S; To et al. 2 005) , and the generation of
recombinant toxin fragments (Liao et al. 2006 ).